The overall goal of this project is to define the molecular basis for the actions of volatile anesthetics, including novel anesthetics and non- immobilizers, on brain receptors. This will be accomplished at two levels. First, glycine, GABAA, and GluR6 (kainate) receptor subunits will be mutated to determine regions critical for anesthetic action. Receptor function and anesthetic sensitivity will be measured electrophysiologically following expression of receptor cDNAs or cRNAs in Xenopus oocytes. This work will involve collaboration with Trudell and Overduin to test predictions form their structural work by studying the function of receptor mutants. The goal of this part of the project is to define the molecular sites responsible for anesthetic modulation of ligand- gated ion channels. Second, the importance of the glycine receptor in anesthesia will be tested by construction of transgenic mice with mutant receptors. Specifically, we will generate mice with mutations in glycine receptor subunits that will eliminate anesthetic action on these receptors while retaining responsiveness to glycine. This will be done by replacing the normal gene with a mutant gene (knock-in) and by making transgenics that over- express the mutant subunit and crossing them with mutant mice lacking this receptor subunit. Mice will be provided to Eger, Sonner, and Kendig for determination of MAC, abolition by test compounds, and electrophysiological studies, respectively. These mutant mice will provide a critical test of the role of glycine receptors in behavioral and electrophysiological actions of anesthetic agents.